Listeria monocytogenes is a food-borne, intracellular pathogen capable of causing gastroenteritis, meningitis, and abortions. Critical for Listeria virulence is its ability to replicate within mammalian cells, and spread from one cell to another through an actin-dependent motility process that allows bacteria to remain within the protective environment of the host cytosol. Cell-cell spread is initiated by the bacterial surface protein ActA, which stimulates the formation of F-actin 'comet tails'that propel Listeria through the cytoplasm. Rocketing bacteria contact the host cell plasma membrane and induce the formation of Listeria-containing protrusions that are engulfed by neighboring cells. Although ActA-mediated actin polymerization is well understood, it remains unclear whether additional bacterial factors promote protrusion formation and intercellular spread by acting after the step of comet tail formation. Our preliminary data indicate that the secreted Listeria virulence protein InlC is needed for efficient cell-cell spread, but is not essential for bacterial-induced F-actin assembly. Importantly, InlC promotes activation (phosphorylation) of host ERM proteins, a family of cytoskeletal regulatory proteins known to participate in Listeria-induced protrusion formation and spread. Moreover, InlC localizes to a subset of comet tails that also contain the ERM protein ezrin. Through a yeast two-hybrid screen, the human adaptor protein Tuba was identified as a ligand of InlC. Like InlC, Tuba co-localizes with ezrin in a subset of comet tails. Tuba contains several functional domains, including SH3 domains that engage ezrin (SH35) or InlC (SH36). Importantly, RNA interference studies indicate that Tuba antagonizes cell-cell spread of Listeria. InlC promotes spreading by relieving the inhibitory effect of Tuba, possibly by impairing binding of human SH36 ligands to Tuba. Based on these results, it seems likely that InlC, Tuba, and ezrin act together to promote cell-cell spread. Tuba might influence spreading by affecting ERM proteins or other effectors. The long-term goal of this project is to understand the molecular mechanism by which InlC promotes intercellular dissemination of Listeria. The Specific Aims in this proposal are directed towards understanding how InlC, ERM proteins, Tuba, and Tuba ligands control cell-cell spread. 1. Determine if InlC and/or Tuba affect formation of F-actin tails or protrusions. 2. Determine if InlC and/or Tuba control spreading through ERM protein phosphorylation. 3. Evaluate the role of human SH36 ligands in intercellular spread of Listeria. The above objectives will be addressed through a variety of approaches, including immunofluorescence microscopy, evaluation of ERM protein phoshorylation through Western blotting, and RNA interference to investigate the function of Tuba and Tuba ligands in cell-cell spread. Listeria monocytogenes is a food-borne, intracellular bacterium that causes serious illnesses leading to meningitis or abortion. Listeria evades the immune system by spreading from one human cell to another, while remaining within the protective environment of the host cytosol. The goal of this research is to identify bacterial and human components that mediate Listeria spreading, and which may be suitable as drug targets.